RESUMO
Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Lectinas/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/química , 1-Desoxinojirimicina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Calreticulina , Sequência de Carboidratos , Clonagem Molecular , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Inibidores de Glicosídeo Hidrolases , Glicosilação , Hexosaminidases/farmacologia , Soros Imunes , Lectinas/efeitos dos fármacos , Lectinas/genética , Dados de Sequência Molecular , Testes de Precipitina , Dobramento de Proteína , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Frações Subcelulares , alfa-GlucosidasesRESUMO
It was found that the digenetic trypanosomatid Endotrypanum schaudinni transferred Man7GlcNAc2 in protein N-glycosylation. Endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were identified as Man7GlcNAc2, Man6GlcNAc2, Rib1Man6GlcNAc2 and/or Gal1Man6GlcNAc2, Man5GlcNAc containing two galactose or ribose units or one of each residues, Rib1Man5GlcNAc2 and Gal1Man5GlcNAc2. The galactoses were in the furanose configuration. Endo-beta-N-acetylglucosaminidase H-resistant glycopeptides that were retained by concanavalin A-Sepharose and eluted with alpha-methylmannoside were found to contain mannose, galactofuranose and ribose units. The presence of galactofuranoses in N-glycoproteins has been reported previously in several monogenetic trypanosomatids but only in one digenetic parasite (Trypanosoma cruzi). This and a recent publication on the structure of Blastocrithidia culicis N-linked oligosaccharides are the first reports on the presence of ribose in eukaryotic glycoconjugates.